Tang Xinyan, Alliston Tamara, Coughlin Dezba, Miller Stephanie, Zhang Nianli, Waldorff Erik I, Ryaby James T, Lotz Jeffrey C
Department of Orthopaedic Surgery, University of California San Francisco, San Francisco, California.
Orthofix Inc., Lewisville, 75056, Texas.
J Orthop Res. 2018 Feb;36(2):778-787. doi: 10.1002/jor.23713. Epub 2017 Oct 17.
Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Pulsed electromagnetic fields (PEMF) are noninvasive biophysical stimulus that has been used extensively in the orthopaedic field for many years. However, the specific cellular responses and mechanisms involved are still unclear. The objective of this study was to assess the time-dependent PEMF effects on pro-inflammatory factor IL-6 expression in disc nucleus pulposus cells using a novel green fluorescence protein (GFP) reporter system. An MS2-tagged GFP reporter system driven by IL-6 promoter was constructed to visualize PEMF treatment effect on IL-6 transcription in single living cells. IL-6-MS2 reporter-labeled cells were treated with IL-1α to mimic the in situ inflammatory environment of degenerative disc while simultaneously exposed to PEMF continuously for 4 h. Time-lapse imaging was recorded using a confocal microscope to track dynamic IL-6 transcription activity that was demonstrated by GFP. Finally, real-time RT-PCR was performed to confirm the imaging data. Live cell imaging demonstrated that pro-inflammatory factor IL-1α significantly promoted IL-6 transcription over time as compared with DMEM basal medium condition. Imaging and PCR data demonstrated that the inductive effect of IL-1α on IL-6 expression could be significantly inhibited by PEMF treatment in a time-dependent manner (early as 2 h of stimulus initiation). Our data suggest that PEMF may have a role in the clinical management of patients with chronic low back pain. Furthermore, this study shows that the MS2-tagged GFP reporter system is a useful tool for visualizing the dynamic events of mechanobiology in musculoskeletal research. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:778-787, 2018.
炎症细胞因子在椎间盘退变的发病机制中起主导作用。脉冲电磁场(PEMF)是一种无创生物物理刺激,多年来已在骨科领域广泛应用。然而,具体的细胞反应和涉及的机制仍不清楚。本研究的目的是使用一种新型绿色荧光蛋白(GFP)报告系统,评估PEMF对椎间盘髓核细胞中促炎因子白细胞介素-6(IL-6)表达的时间依赖性影响。构建了由IL-6启动子驱动的MS2标签GFP报告系统,以可视化PEMF对单个活细胞中IL-6转录的治疗效果。用IL-1α处理IL-6-MS2报告基因标记的细胞,以模拟退变椎间盘的原位炎症环境,同时连续4小时暴露于PEMF。使用共聚焦显微镜记录延时成像,以追踪由GFP证明的动态IL-6转录活性。最后,进行实时逆转录聚合酶链反应(RT-PCR)以确认成像数据。活细胞成像显示,与DMEM基础培养基条件相比,促炎因子IL-1α随时间显著促进IL-6转录。成像和PCR数据表明,PEMF处理可时间依赖性地显著抑制IL-1α对IL-6表达的诱导作用(早在刺激开始2小时)。我们的数据表明,PEMF可能在慢性下腰痛患者的临床管理中发挥作用。此外,本研究表明,MS2标签GFP报告系统是在肌肉骨骼研究中可视化力学生物学动态事件的有用工具。©2017作者。《骨科研究杂志》®由威利期刊公司代表骨科研究协会出版。《骨科研究杂志》35:778 - 787,2018年。
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